Receptors localized to cilia are fundamental to our senses of sight and smell, and receptors on primary cilia are used by most vertebrate cells to sense the extracellular environment. These organelles allow cells to coordinate their proliferation, differentiation and physiology with external conditions and are of great importance during development and in tissue homeostasis. In mice, mutations that block the formation of cilia cause embryonic death at mid gestation. Less severe mutations cause pleiotropic phenotypes including cystic kidney disease and retinal degeneration, among others. In humans, ciliary defects cause a diverse set of diseases that have come to be known as the ciliopathies. These include polycystic kidney disease and retinal degeneration as well as syndromes such as Bardet-Biedl, Jeune, Meckel Gruber, and Senior Loken that often combine kidney disease and blindness with other phenotypes like heart defects, obesity and skeletal abnormalities. Primary cilia are assembled via the process of intraflagellar transport (IFT). During IFT, large protein complexes called IFT particles carry ciliary precursors from their site of synthesis in the cell body to their site of assembly at the tip of the cilium. We are interested in the function of the IFT particle proteins and have focused on the IFT20 subunit. This protein is unique in that it is found at the Golgi complex in addition to the cilium. We have shown that IFT20 is required for ciliary assembly and that it cycles between the Golgi apparatus and the cilium. At the Golgi membrane, IFT20 is complexed with the golgin protein GMAP210. Partial knockdowns of IFT20 or complete loss of GMAP210 do not completely block ciliary assembly but reduce the ciliary levels of the membrane protein polycystin-2. This makes IFT20 and associated proteins good candidates for trafficking membrane proteins to the ciliary compartment. Understanding the mechanisms of ciliary membrane protein transport will provide insight into the etiology of many ciliary diseases since the sensory functions of cilia depend on the proper localization of membrane proteins. In this proposal, we will address the function of IFT20 by studying the role of the GMAP210-IFT20 complex in trafficking ciliary membrane proteins. We will also identify new IFT20-interacting proteins. We will address the importance of these proteins by RNAi studies in cultured mammalian cells and by mutant mouse studies. In addition to studying IFT and IFT20-interacting proteins, we will investigate the function of a ciliary targeting sequence to better understand how receptors are localized to primary cilia. To do this, we identified an 18 residue peptide from a ciliary membrane protein that is capable of directing exogenous proteins to the cilium. We have shown that this peptide binds to several proteins implicated in ciliary assembly and protein trafficking. We are testing to see if these proteins directly interact with the ciliary targeting sequence and if they are required for trafficking proteins to the ciliary membrane. PUBLIC HEALTH RELEVANCE: Severe defects in the primary cilium cause prenatal lethality while less severe defects result in a variety of diseases including polycystic kidney disease and blindness along with a group of syndromes called the ciliopathies. These syndromes, which include Bardet-Biedl, Meckel Gruber and Jeune Syndromes, often present with cystic kidney disease and blindness along with a variety of symptoms including obesity, mental retardation, and left-right asymmetry defects of the heart and body. This proposal seeks to understand the mechanism by which cilia are assembled and ultimately drive the identification of a treatment or cure for these diseases.